S-Adenosylhomocysteine hydrolase is the only enzyme in mammalian cells for the removal of adenosylhomocysteine, the end-product of biological transmethylation reactions. For this reason, the enzyme is critical for th regulation of adenosylmethionine-dependent methylations. We have used several approaches to investigate structure/function relationships of AdoHc hydrolase. We have cloned and expressed the cDNA for the enzymes from rat liver, D. discoideum, and Rhodobacter capsulatus. The amino acid sequences are highly conserved between species suggesting that much of the sequence i required for enzyme function. Human and bacterial amino acid sequences hav 64% sequence identity; to our knowledge this is the highest level of conservation reported between human and prokaryotic enzymes. The putative NAD binding site has been identified by homology to several dehydrogenases and by site-directed mutagenesis of specific amino acids within this region Investigation of the inactivation of the rat liver enzyme by the site-specific reagent, p-fluorosulfonylbenzoyladenosine, yielded data that support the role of a specific cysteine (cysteine 78) in enzyme function. During inactivation of the enzyme a disulfide between cysteine 78 and cysteine 112 is formed that can be reduced with a thiol to reactivate the enzyme. When cysteine 112 is mutated to alanine, an enzyme with nearly identical kinetic properties is obtained; but upon inactivation by p- fluorosulfonylbenzoyladenosine, cysteine 78 forms a disulfide with cysteine 52. Disulfide formation in the wild-type and mutant enzymes suggests that cysteines 52, 78 and 112 may be near each other in the three dimensional structure of the protein. We have found a class of organisms that metabolize adenosylhomocysteine by deamination. The Km for AdoHcy is in the micromolar range suggesting that AdoHcy may be one of the normal substrates. The enzyme is inhibited by coformycin and cells treated with coformycin accumulate AdoHcy.